The variable domain of heavy-chain only antibodies found in members of the camelid family represents the smallest naturally occurring functional domain of the antibody molecule. These variable domains, termed VHH, have the same antigen binding capability as full-length antibodies, yet are typically around 15 kDa in size. When cloned and purified as monomeric domains, VHH demonstrate remarkable stability under a wide range of denaturing, temperature, and pH conditions. VHHs exhibit increased solubility compared to full-length antibodies or other antibody fragments, and very high expression levels have been achieved in E. coli, yeast, and tobacco expression systems. Due to a high degree of sequence homology between camelid and other mammalian variable domains, VHH have been shown not to be immunogenic in mice. As a result of their small size, VHH have enhanced tissue penetration, and an extended CDR3 loop allows VHH access to cryptic epitopes in enzymatically active sites that are unavailable for binding by full length antibodies.
Given their unique combination of characteristics, VHH have been promoted as promising biomedical tools. A myriad of VHH have been successfully developed for diverse purposes including diagnostics, imaging, and biochemical and therapeutic applications. In terms of the diversity of pathogens that have been targeted thus far, VHH directed against viruses, bacteria, protozoa, and fungi have all been identified. VHH can act as a monomeric domain, or they can be expressed in a multivalent context to increase avidity and activity. Additionally, bispecific VHH can be assembled that bind different epitopes, which can in some cases dramatically increasing neutralization efficacy.
HSV-2 is one of the most prevalent sexually transmitted infections (STIs) in the world, and recent estimates indicate that roughly 16% of people ages 15-49 worldwide are infected. There has been great interest in the development of a prophylactic vaccine to prevent HSV-2 infection over the past several decades, but unfortunately, an effective one has yet to be developed.
Human immunodeficiency virus type I (HIV-1) is also a sexually transmitted infection, and has contributed to an estimated 40 million deaths since it was first recognized in 1981. Currently, over 30 million people worldwide are living with the virus. The development of effective HIV-1 vaccine immunogens that can elicit high titer, potent, and broadly neutralizing antibodies (bnAbs) remains a major challenge.
Entry of HIV-1 into target cells is mediated by binding of highly conserved epitopes on HIV envelope glycoproteins (Env) to a primary cell-surface receptor CD4. Binding of Env to CD4 initiates a series of conformational changes of the Env structure, leading to exposure and/or formation of coreceptor binding sites that are recognized by cell surface co-receptors (e.g. chemokine receptors CCR5 or CXCR4). Since HIV-1 was first discovered more than two decades ago, conventional vaccine strategies have failed to develop effective vaccine candidates that can elicit potent broadly cross-reactive HIV-1-neutralizing antibodies. There continues to be a pressing need for novel HIV vaccine strategies and compositions that can control the spread of HIV/AIDS pandemic.
A microbicide is a substance that can be applied to mucosal surfaces, including the vagina and rectum, to prevent infection with an STI. A significant public health goal has been to try develop a successful microbicide against HSV-2 and HIV-1, including vaginal delivery of antiviral drugs, antibody-based strategies, and small-interfering RNAs. It has been demonstrated that vaginally applied monoclonal antibodies and single chain antibody variable fragments (scFv) directed against gD2 protect against HSV-2 infection in animal models. The issue of how to vaginally deliver a neutralizing antibody against HSV2 or HIV-1 without the direct application of the antibody immediately prior to sexual intercourse has yet to be resolved, however. Furthermore, the current methods of production of monoclonal antibodies and scFvs can be cost-prohibitive to scale up, as antibodies are complex molecules with multiple protein chains that are not easily purified and assembled.
As a result, there still exists an unmet need for alternative strategies to prevent transmission, including the development of an effective microbicide using means other than scFvs.